Infectious diseases contribute significantly to human morbidity and mortality. Many pathogens that cause disease in humans, exhibit a very narrow host range, which poses challenges both for study and for developing effective clinical therapies. We will focus our studies on hepatitis C virus (HCV), an archetypal hepatotropic virus with a narrow host range and derive cell-intrinsic immune response patterns that impact infection outcome across species. Eighty percent of HCV infections result in chronic infection and can lead to cirrhosis and hepatocellular carcinoma. HCV has an incompletely defined host tropism limited almost exclusively to humans and chimpanzees. We have previously demonstrated that differences (between human and mouse) between the amino acid sequences of two host factors, CD81 and occludin (OCLN) block HCV uptake in mice. While HCV assembly and release is supported in hepatocytes across a broad variety of species ranging from humans to rodents, antiviral innate responses appear to limit HCV RNA replication in non-human species. We hypothesize that species-specific differences in the kinetics, diversity and magnitude of antiviral gene products influence viral tropism. Here, we propose to develop and utilize a novel platform to systematically define such species-specific response patterns contributing to infection outcome. We aim to capitalize on a collection of dermal fibroblasts from non-human primate species to generate induced pluripotent stem cells (iPSCs) and differentiate these simian iPSCs in a stepwise manner to hepatocyte like cells (HLCs). We will characterize and compare viral infections in HLCs derived from human, chimpanzee, bonobo, gorilla, orangutan and rhesus monkey specimens. Orthologs of essential host factors critical for supporting the viral life-cycle are largely conserved across these species and differences in susceptibility can likely be attributed to differences in antiviral response patterns and/or HCV's inability to antagonize cell-intrinsic defenses. We will couple a HCV infection-dependent fluorescence relocalization (HDFR) reporter with laser capture microdissection (LCM) to isolate infected cells from HCV infected HLCs from different species and subject them to transcriptomic profiling. By comparing and contrasting data from species with high and low infection frequencies, we will be able to determine species-specific signatures associated with effective antiviral control. The success of this collaborative project depends on the complementary expertise in HCV virology and viral tropism (Ploss lab) and stem cell and hepatocyte biology (Schwartz lab). We are convinced that this systems virology approach will provide rich insights into evolutionary conservation of antiviral response patterns and shed light on the impact of antiviral immunity to restrict species tropism. Since many of the primate species for which we collect materials are endangered and cannot be used (anymore) in biomedical research, the emerging iPSC repository we will build will also be an enabling platform for other researchers needing access to usually inaccessible primate species/tissues.